| Objective:The purpose of this study is to establish an efficient, simple and reproducible preparative isolation method of morellic acid(MA), then the isolated MA were pharmacy modified in order to enhance its anti-tumor activity and to improve their pharmacokinetic characteristics.Methods:Ethanol ultrasonic extraction method was used for extraction of resin of garcinia hanburyi. Medium-pressure preparative chromatographic was used for isolation of MA,and the orthogonal experiment was conducted to optimize the extraction and isolation processes. High-pressure preparative chromatography systems was used to obtain the purified MA, and MS, IR, 1H-NMR and 13C-NMR was used for the structural confirmation of MA purified samples. A emulsion evaporation-low temperature curing method was used for preparation of MA nanostructure lipid carriers(MA-NLC), and the single factor test and orthogonal test was conducted to select a relatively suitable preparation process. Characterization of MA-NLC was conducted for the quality control.MA-NLC lyophilized powder was prepared by freeze-drying method, and the quality of MA-NLC lyophilized powder was also evaluated. MTT assay was used to compare cytotoxic of MA solution and MA-NLC against human gastric cancer MGC-803 cells and human colon cancer HCT-116 cells in order to evaluate the in vitro antitumor activity of MA-NLC. HPLC analysis method MA in rat plasma was established and the over time drug concentration was determined to observe the pharmacokinetics dynamic behaviors of MA-solution and MA-NLC. Pharmacokinetic software was used to obtain the main pharmacokinetic parameters.Results:The purity of MA sample prepared by the established efficient, simple and reproducible preparative separation method can reach 98%, and isolated sample was identified as morellic acid(MA). In the study of preparation of MA-NLC,morphology of MA-NLC showed that the prepared MA-NLC was spherical and without adhesion. After the single factor and orthogonal experiment, encapsulation efficiency of prepared MA-NLC can reach 78.26%. The average particle size was 165.5nm. The average zeta potential was-21.85 mv. The DSC analysis showed that the prescription of each component forms a new phase. The stability test results showed that a freshly prepared colloidal solution of MA-NLC was stable under 4 ℃ within 60 d, while at25 ℃, it was basically stable within 30 d. The results of in vitro release test showed that MA-NLC has significant slow release effect. The release time is longer than the MA solution of 6.86 times. Physical stability of MA-NLC freeze-dried powder prepared by freeze-dry significantly improved,which was able to maintain the basic stability and well dispersed within 60 d at 4 ℃ and 25 ℃. There was no significant difference in other characterization results compared with non-lyophilized sample. MTT colorimetric test results showed that the in vitro anti-tumor activity of MA-NLC was significantly better than the MA-solution,half IC50 value of MA solution and MA-NLC against human gastric cancer MGC-803 cells and human colon cancer HCT-116 cells were11.693μg?m L-1and 0.395μg?m L-1, respectively. The in vitro anti-tumor effect of MA-NLC against the two cell lines were increased 2.04 times and 3.43 times compared to MA solution group. Pharmacokinetic results showed that, the circulation time of MA-NLC in vivo was longer than MA solution. The t1/2α of MA-solution and MA-NLC were 2.9741 min and 17.1857 min, respectively, and t1/2β were 21.0634 min and167.0535 min, respectively Cmax of MA-solution and MA-NLC were 0.3543μg?m L-1and 0.2915μg?m L-1, respectively. AUC 0-t of MA-solution and MA-NLC were 3.8168μg/m L?min and 18.9058μg/m L?min respectively. AUC0-inf of MA-solution and MA-NLC were 5.1520μg/m L?min and 20.1106μg/m L?min, respectively. MRT of MA-solution and MA-NLC were 23.9354 min and 191.3139 min, respectively.Conclusion:MA preparative separation method which was established in this paper has a certain operability and usability. Preparation of MA-NLC, the in vitro pharmacologicalstudy and pharmacokinetics study will provide new ideas in future investigations of MA... |
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